Not Applicable
Not Applicable
This invention pertains to the fields of molecular biology and oncology. In particular, this invention provides a novel two-hybrid system to screen for agents that modulate the ability of a cell to degrade a metabolic product (e.g. certain cancer cells) or to selectively kill a cell that has a defect in its ability to degrade a metabolic product.
A wide variety of diseases are characterized by the abnormal accumulation of one or more metabolic products. Such diseases, sometimes referred to as xe2x80x9cstorage diseasesxe2x80x9d are typically caused by the increased accumulation of metabolic products (e.g., lipids, proteins, complex carbohydrates, etc.) due to either the inactivity of an enzyme that degrades the products or the hyperactivity of an enzyme that creates the products. Storage disease include but are not limited to glycogen storage disease 1, GM1 gangliosidoses, MPS IV B (Morquio B), GM2 gangliosidoses (O, B, AB, B1 variants), Niemann-Pick disease (A, B, and C), Metachromatic leukodystrophy (arylsulfatase A and SAP-1 deficient), Krabbe disease, Fabry disease, Gaucher disease, Farber disease, Wolman disease (cholesterol ester storage disease), MPS I (Hurler and Scheie syndromes), MPS II (Hunter syndrome), MPS III A, C, and D (Sanfilippo A, C, and D), PS III B (Sanfilippo B), MPS IV A (Morquio A), MPS VI (Maroteaux-Lamy syndrome) MPS VII (beta-glucuronidase deficiency), Multiple sulfatase deficiency, Mucolipidosis I (Sialidosis), Mucolipidosis II and III, alpha-Mannosidosis, beta-Mannosidosis, Fucosidosis, Sialic acid storage disease, Galactosialidosis, and Aspartylglucosaminuria Cystinosis.
Historically, storage diseases have been treated by supplementing the xe2x80x9cmissingxe2x80x9d enzymatic activity. Thus, for example, Gaucher""s disease can be treated by use of a glucocerebrosidase targeted to spleen cells. Similarly, superoxide dismutase can be targeted to the liver as an anti-oxidant, and so forth.
Such approaches typically have seen limited success. Often the xe2x80x9ctherapeutic agentxe2x80x9d must be specifically targeted to a particular organ or tissue. In addition consistent delivery of the targeted therapeutic agent at physiologically relevant concentrations has proven difficult. In addition, the identification of viable therapeutic agents has proven difficult.
This invention provides novel approaches to the treatment (e.g. amelioration of one or more symptoms) of pathological states characterized by the undesired accumulation of one or more metabolic products. In addiction, this invention provides effective systems in which to screen for agents that modulate the ability of a cell to accumulate and/or degrade a metabolic product.
In preferred embodiments, this invention utilizes a two-hybrid system to screen for agents that modulate the ability of a cell to degrade or to accumulate a metabolic product or to selectively kill a cell or to selectively express a gene or cDNA in a cell that has a defect in its ability to degrade or to accumulate a metabolic product. In one such embodiment, this invention provides a method of screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product. The method involves providing a mammalian cell comprising a nucleic acid encoding a peptide binding domain and an effector gene; a first chimeric protein comprising a nucleic acid binding domain that binds the peptide binding domain attached to the metabolic product or to a ligand that binds to the metabolic product; and a second chimeric protein comprising an expression control protein attached to the metabolic product or to the ligand that binds to the metabolic product such that when the first chimeric protein comprises the metabolic product, said second chimeric protein comprises the ligand and when the first chimeric protein comprises the ligand, said second chimeric protein comprises the metabolic product. The cell is contacted with one or more test agent(s) and alteration of expression of the effector gene is detected wherein a difference in the expression of the effector gene in the test cell, as compared to a control cell contacted with a lower concentration of test agent or no test agent indicates that the test agent(s) modulate the ability of said cell to accumulate or degrade the metabolic product.
In particularly preferred embodiments, the expression control protein is a transactivator (e.g. VP16) or a repressor. Particularly preferred nucleic acid binding proteins include, but are not limited to GAL-4, and GAL-4-Y. In certain embodiments, preferred effectors include, but are not limited to, a reporter gene (e.g., chloramphenicol acetyl transferase (CAT), luciferase, b-galactosidase (b-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), green fluorescent protein (GFP), etc.), a cytotoxin (e.g., thymidine kinase, pseudomonas exotoxin, diphtheria toxin, ricin, abrin, etc.), and/or an apoptosis inducing (e.g. P53, P73, Bax, Bad, FADD, a caspase gene, (ect). In various embodiments, the ligand and metabolic product respectively include, but are not limited to a beta-catenin and a Tcf, a NF-kB and I-kB, a P53 and MDM2, a receptor and its heteromelic receptor partner.
The first and/or the second chimeric protein(s) are expressed from a nucleic acid in the cell (e.g. under control of an inducible (e.g. ecdysone promoter), tissue-specific, or constitutive promoter), or the first and/or the second chimeric protein is a protein transported into the cell, e.g. by using a protein comprising an HIV TAT domain.
Preferred cells for the practice of the methods of this invention include, but are not limited to SW480, SW48, DLD-1, HCT-116, HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, and/or Jarkat cell.
In one particularly preferred embodiment the nucleic acid encodes a GAL-4 binding site, the effector gene is a reporter gene, the first chimeric protein comprises a GAL-4 nucleic acid binding protein and a beta catenin or a Tcf; and the second chimeric protein comprises a VP-16 and beta catenin or a Tcf. One particularly preferred Tcf is Tcf4.
In certain embodiments, the cell further comprises a second nucleic acid encoding the ligand or metabolic product operably linked to an inducible promoter (e.g. an ecdysone promoter).
In another embodiment, this invention provides a method of selectively expressing an effector gene in a cell that accumulates or degrades a metabolic product. The method involves providing a cell comprising a nucleic acid encoding a peptide binding site and an effector gene, a first chimeric protein comprising a nucleic acid binding protein that binds the peptide binding domain where the nucleic acid binding protein is attached to the metabolic product or to a ligand that binds to said metabolic product, and a second chimeric protein comprising an expression control protein attached to the metabolic product or to a ligand that binds to the metabolic product such that when said first chimeric protein comprises the metabolic product, said second chimeric protein comprises the ligand and when the first chimeric protein comprises the ligand, said second chimeric protein comprises the metabolic product; whereby the cell, in the absence of the ability to degrade the metabolic product or the ligand that binds the metabolic product activates or represses expression of said effector gene. A preferred expression control protein is a transactivator (e.g. VP16) or a repressor. Preferred nucleic acid binding proteins, effector genes, ligands and metabolic products include, but are not limited to those described above.
The first and/or the second chimeric protein(s) are expressed from a nucleic acid in the cell (e.g., under control of an inducible (e.g. ecdysone promoter), tissue-specific, or constitutive promoter), or the first and/or the second chimeric protein is a protein transported into the cell, e.g. by using a protein comprising an HIV TAT domain.
Preferred cells for the practice of the methods of this invention include, but are not limited to SW480, SW48, DLD-1, HCT-116, HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, and/or Jarkat cell.
In one particularly preferred embodiment the nucleic acid encodes a GAL-4 binding site, the effector gene is a reporter gene, the first chimeric protein comprises a GAL-4 nucleic acid binding protein and a beta catenin or a Tcf; and the second chimeric protein comprises a VP-16 and beta catenin or a Tcf. One particularly preferred Tcf is Tcf4.
This invention also provides methods of selectively killing a cell that accumulates a metabolic product (e.g. certain cancer cells). The methods involve transfecting the cell with a nucleic acid encoding a peptide binding site and an effector that is a cytotoxin or an apoptosis-inducing gene; introducing into the cell a first chimeric protein comprising a nucleic acid binding protein that binds the peptide binding domain where said nucleic acid binding protein is attached to said metabolic product or to a ligand that binds to said metabolic product; and introducing into the cell a second chimeric protein comprising a transactivator (e.g. VP16) attached to said metabolic product or to said ligand that binds to said metabolic product, such that when said first chimeric protein comprises said metabolic product, said second chimeric protein comprises said ligand and when said first chimeric protein comprises said ligand, said second chimeric protein comprises said metabolic product. Preferred nucleic acid binding proteins include, but are not limited to, GAL-4 and GAL-4-Y. In one preferred embodiment, the effector is a cytotoxin (e.g. thymidine kinase, pseudomonas exotoxin, diphtheria toxin, ricin, abrin, etc.). In certain preferred embodiments, the effector is an apoptosis-inducing gene (e.g. P53, P73, Bax, Bad, FADD, a caspase (e.g. Casp3, Casp9, Apaf1, etc.), etc.). In various embodiments, the ligand and metabolic product respectively include, but are not limited to a beta-catenin and a Tcf, a NF-kB and I-kB, a P53 and MDM2, a receptor and its heteromelic receptor partner.
The first and/or the second chimeric protein(s) are expressed from a nucleic acid in the cell (e.g. under control of an inducible (e.g. ecdysone promoter), tissue-specific, or constitutive promoter), or the first and/or the second chimeric protein is a protein transported into the cell, e.g. by using a protein comprising an HIV TAT domain. Suitable cells include, but are not limited to SW480, SW48, DLD-1, HCT-116, HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, Jarkat cells, etc. In one embodiment, the nucleic acid encodes a GAL-4 binding site, the first chimeric protein comprises a GAL-4 nucleic acid binding protein and a beta catenin or a Tcf, and the second chimeric protein comprises a VP-16 and beta catenin or a Tcf (e.g. Tcf4).
In still another embodiment this invention provides vertebrate, preferably mammalian cells comprising one or more constructs of this invention. In one embodiment the cell is it cell comprising a nucleic acid encoding a peptide binding site and an effector gene, a first chimeric protein comprising a nucleic acid binding protein that hinds the peptide binding domain where the nucleic acid binding protein is attached to the metabolic product or to a ligand that binds to the metabolic product; and a second chimeric protein comprising an expression control protein attached to the metabolic product or to the ligand that binds to the metabolic product such that when the first chimeric protein comprises said metabolic product, the second chimeric protein comprises the ligand and when said first chimeric protein comprises the ligand, the second chimeric protein comprises the metabolic product. Expression control proteins, first and second nucleic acids, effector genes, nucleic acid binding domains include, but are not limited to those described above.
In still another embodiment this invention provides a nucleic acid selected from the group consisting of: a nucleic acid encoding a chimeric protein comprising a nucleic acid binding domain attached to a Tcf or to a beta catenin, and a nucleic acid encoding a transactivator attached to a beta catenin or to a Tcf. Particularly preferred nucleic acids include a nucleic acid encoding a nucleic acid binding protein attached to a Tcf4, a nucleic acid encoding a nucleic acid binding protein attached to a beta catenin, a nucleic acid encoding a Tcf4 attached to a transactivator, and a nucleic acid encoding a beta catenin attached to a transactivator (e.g. VP16). Preferred nucleic acids include DNAs or RNAs and in certain embodiments, the nucleic acid comprises a vector.
In still another embodiment this invention provides a kit for screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product. Preferred kits comprise a container containing a mammalian cell comprising a nucleic acid encoding a protein binding site and an effector gene; a first chimeric protein comprising a nucleic acid binding protein that binds said peptide binding domain attached to said metabolic product or to a ligand that binds to said metabolic product; a second chimeric protein comprising an expression control protein attached to said metabolic product or to said ligand that binds to said metabolic product such that when said first chimeric protein comprises said metabolic product, said second chimeric protein comprises said ligand and when said first chimeric protein comprises said ligand, said second chimeric protein comprises said metabolic product.
In another embodiment, the kit a container containing a nucleic acid as described herein.
Also provided are kits for selectively killing a cell. Such kits include a container containing a two-hybrid system component selected from the group consisting of one or more nucleic acids as described herein, and/or one or two chimeric proteins as described herein.
A xe2x80x9cchimeric moleculexe2x80x9d is a molecule comprising two or more molecules typically found separately in their native state that are joined together typically through one or more covalent bonds. The molecules may be directly joined or joined through a linker. Where the molecules are both polypeptides they may be joined through peptide bond or a peptide linker forming a fusion protein.
A xe2x80x9cfusion proteinxe2x80x9d refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein halving a single contiguous polypeptide backbone.
A xe2x80x9cspacerxe2x80x9d or xe2x80x9clinkerxe2x80x9d as used in reference to a fusion protein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.
A xe2x80x9cspacerxe2x80x9d or xe2x80x9clinkerxe2x80x9d as used in reference to a chemically conjugated chimeric molecule refers to any molecule that links/joins the constituent molecules of the chemically conjugated chimeric molecule.
The xe2x80x9cmetabolic productxe2x80x9d refers to a molecule formed as an intermediate or an end product of a metabolic pathway. The term may also include such products as they may be subsequently modified by member of a different pathway in a cell. When used as a domain of a chimeric molecule in this invention the metabolic product may be a full-length/full-size metabolic product or a fragment thereof sufficient to be specifically bound by another molecule.
The phrase xe2x80x9ckill or inhibit a cellxe2x80x9d refers to the death of a cell or a decrease in cellular growth and/or proliferation.
The term xe2x80x9cspecifically bindsxe2x80x9d, when referring to the interaction of a nucleic acid binding protein and a nucleic acid binding site or two proteins or other binding pairs refers to a binding reaction which is determinative of the presence of the one or other member of the binding pair in the presence of a heterogeneous population of molecules (e.g., proteins and other biologics). Thus, for example, in the case of a receptor/ligand binding pair the ligand would specifically and/or preferentially select its receptor from a complex mixture of molecules, or vice versa. An enzyme would specifically bind to its substrate, etc. The binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, covalent interactions, hydrophobic interactions, van der Waals interactions, etc.
The terms xe2x80x9cbinding partnerxe2x80x9d, or a member of a xe2x80x9cbinding pairxe2x80x9d, or xe2x80x9ccognate ligandxe2x80x9d refers to molecules that specifically bind other molecules to form a binding complex such as antibody/antigen, lectin/carbohydrate, nucleic acid/nucleic acid, receptor/receptor ligand (e.g. IL-4 receptor and IL-4), avidin/biotin, etc.
The term ligand is used to refer to a molecule that specifically binds to another molecule. Commonly a ligand is a soluble molecule, e.g. a hormone or cytokine, that binds to a receptor. The decision as to which member of a binding pair is the ligand and which the xe2x80x9creceptorxe2x80x9d is often a little arbitrary when the broader sense of receptor is used (e.g., where there is no implication of transduction of signal). In these cases, typically the smaller of the two members of the binding pair is called the ligand. Thus, in a lectin-sugar interaction, the sugar would be the ligand (even if it is attached to a much larger molecule, recognition is of the saccharide).
The term xe2x80x9ctwo-hybrid systemxe2x80x9d refers to a system comprising two chimeric molecules one of which bears a nucleic acid binding region, the other of which bears an expression control element (e.g. a transactivation or repressor domain). The molecules further a cognate binding pair such that one chimeric molecule is capable of specifically binding to the other chimeric molecule. The two-hybrid system further comprises a nucleic acid encoding a protein binding site that is specifically bound by the protein binding domain on the chimeric molecule thereby anchoring the chimeric molecule to the nucleic acid. The domain of the chimeric molecule recognizes and binds to its cognate binding partner on the second chimeric molecule thereby recruiting that molecule to the nucleic acid whereby the expression control element alters (e.g. activates) expression of a gene or cDNA comprising the underlying nucleic acid.
The phrase xe2x80x9cone or more components of the two-hybrid systems of this intentionxe2x80x9d refers to either or both of the chimeric molecules comprising the two-hybrid system and/or the nucleic acid(s) encoding them and/or the nucleic acid comprising the protein recognition site and effector.
xe2x80x9cTransfectionxe2x80x9d is used herein to mean the delivery of expressible nucleic acid to a target cell, such that the target cell is rendered capable of expressing said nucleic acid. It will be understood that the term xe2x80x9cnucleic acidxe2x80x9d includes both DNA and RNA without regard to molecular weight, and the term xe2x80x9cexpressionxe2x80x9d means any manifestation of the functional presence of the nucleic acid within the cell, including without limitation, both transient expression and stable expression.
xe2x80x9cDeliveryxe2x80x9d is used to denote a process by which a desired compound is transferred to a target cell such that the desired compound is ultimately located inside the target cell or in, or on the target cell membrane. In many uses of the compounds of the invention, the desired compound is not readily taken up by the target cell and delivery via lipid aggregates is a means for getting the desired compound into the cell. In certain uses, especially under in vivo conditions, delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
The terms xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9coligonucleotidexe2x80x9d refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acid Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419), phosphorodthioate (Mag et al. (1991) Nucleic Acids Res. 19:1437, and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111: 2321, O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et at. (1996) Nature, 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside and Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, xe2x80x9cCarbohydrate Modifications in Antisense Researchxe2x80x9d, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic and Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995). Chem. Soc. Rev; pp 169-176). Several nucleic acid analogs are described in Rawls, C and E News Jun. 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
A xe2x80x9crecombinant expression cassettexe2x80x9d or simply an xe2x80x9cexpression cassettexe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a gene or cDNA in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein.
The terms xe2x80x9cgenexe2x80x9d and xe2x80x9ccDNAxe2x80x9d are typically used interchangeably herein. Thus, for example, a reporter gene can comprise a genomic nucleic acid (e.g. comprising introns and exons) or a cDNA. In certain instances, the terms DNA and cDNA will not be used interchangeably. This may he determined from the context of the usage.
An xe2x80x9capoptosis inducing genexe2x80x9d refers to a gene or cDNA implicated in apoptosis. Preferred apoptosis genes induce or increase apoptosis when they are activated. Apoptosis inducing genes include, but are not limited to P53, P73, Bax, Bad, FADD, caspases, etc.).
A method of xe2x80x9cselectively killingxe2x80x9d refers to a method that induces death of a particular cell type more frequently and/or sooner than the death of a different cell type (e.g. a cell lacking the particular distinguishing feature(s)) of the xe2x80x9ctargetedxe2x80x9d cell.
The term xe2x80x9ctransactivatorxe2x80x9d refers to a molecule that induces transcription and/or upregulates transcription of a gene or cDNA. The transactivator may be a complete xe2x80x9cnativexe2x80x9d molecule or a domain of a molecule that is capable of inducing and/or upregulating transcription of a gene or cDNA.
An xe2x80x9cexpression control domainxe2x80x9d or xe2x80x9cexpression control proteinxe2x80x9d is typically a polypeptide that alters the expression of a gene or cDNA. Typical expression control proteins are transactivators (that upregulate expression of a gene or cDNA) or repressors (that downregulate the expression of a gene or cDNA).
A xe2x80x9ccytotoxinxe2x80x9d is an agent (e.g. a protein) that kills a cell.
An xe2x80x9capoptosis genexe2x80x9d refers to a gene whose expression is involved in an apoptosis pathway. Such apoptosis genes include, but are not limited to P53, P73, Bax, Bad, FADD, a a caspase gene, etc.
The term xe2x80x9ctest agentxe2x80x9d refers to any agent that is to be screened for a desired activity. The xe2x80x9ctest compositionxe2x80x9d can be any molecule or mixture of molecules, optionally in a suitable carrier.
The term xe2x80x9csmall organic moleculexe2x80x9d typically refers to molecules of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
An xe2x80x9ceffector moleculexe2x80x9d is any molecule whose expression is to be controlled by the two-hybrid system of this invention. Suitable effector molecules include, but are not limited to cytotoxins, reporter genes, enzymes, apoptosis genes, and the like.
xe2x80x9cReporter genesxe2x80x9d are genes or cDNAs that express an easily assayable (detectable and/or quantifiable) product. Detection of the assayable product indicates the expression and/or level of expression of the reporter gene. Reporter genes are well known to those of skill in the art. They include, but are not limited to, genes expressing bacterial chloramphenicol acetyl transferase (CAT), beta-galactosidase (xcex2-gal), green fluorescent protein (GFP) and other fluorescent protein, various bacterial luciferase genes, e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and Xenorhabdus luminescens, the firefly luciferase gene FFlux, and the like.
The term xe2x80x9cmodulatexe2x80x9d when used with reference to the ability of a cell to accumulate or to degrade a metabolic product.
The terms xe2x80x9cpeptide binding sitexe2x80x9d or xe2x80x9cprotein binding sitexe2x80x9d refer to a nucleic acid sequence that is recognized and/or bound by a nucleic acid binding protein (e.g. a DNA binding protein).
A xe2x80x9cnucleic acid binding domainxe2x80x9d refers to a protein or a region of a protein that recognizes and/or binds to a nucleic acid. A nucleic acid binding protein (e.g., a DNA binding protein) can be a full-length nucleic acid binding protein or a fragment thereof that binds to a nucleic acid.
The term xe2x80x9ckitxe2x80x9d refers to a collection of materials, more preferably a packaged collection of materials (preferably related materials) to perform a particular function (e.g. to run a screening assay, to express a protein, to culture a cell, etc.). A kit may optionally comprise instructional materials describing the use of the materials present in the kit.